Nucleotide sequence encoding a modulator of nf-kb

ABSTRACT

The present invention relates to nucleotide sequences encoding a modulator of NF-κB, and to the polypeptides encoded by the nucleotide sequences. In particular, the invention relates to nucleotide sequences and the polypeptides encoded thereby, wherein the polypeptides are involved in the response to NF-κB-activating stimuli, including HTLV-1 Tax, LPS, PMA and IL-1. The invention also relates to antibodies to the modulator of NF-κB, methods of detecting modulator of NF-κB using the antibodies, methods of treatment associated with NF-κB activation and to methods of identifying compounds which modulate the activity of the modulator of NF-κB.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to nucleotide sequences encoding amodulator of NF-κB, and to the polypeptides encoded by the nucleotidesequences. In particular, the invention relates to nucleotide sequencesand the polypeptides encoded thereby, wherein the polypeptides areinvolved in the response to NF-κB activating stimuli, including HTLV-1Tax, LPS, PMA and IL-1.

2. Discussion of the Background

The Rel/NF-κB family of transcription factors plays important roles inimmune and stress responses, in inflammation, in apoptosis, andregulates the expression of numerous cellular and viral genes (forrecent reviews, see Baldwin, 1996; Verma et al., 1995; May and Ghosh,1998). The NF-κB activity is composed of homo- or heterodimers ofrelated proteins that share a conserved DNA-binding and dimerizationdomain called the Rel Homology Domain. In most cell types, NF-κB issequestered in the cytoplasm bound to inhibitory proteins called IκB-α,IκB-β and IκB-ε. In response to diverse stimuli, including inflammatorycytokines, mitogens, bacterial lipopolysaccharide (LPS), or some viralproducts, active NF-κB is released and translocated to the nucleus as aresult of the proteolytic degradation of IκB proteins. Phosphorylationof IκBα on Ser 32 and 36 targets the molecule for degradation by theubiquitin-26S proteasome pathway. While the processes leading to thedegradation of the IκB proteins are relatively well understood, themechanism by which a variety of distinct signals initiated from the cellmembrane are transduced to their common targets, the IκB proteins,remains to be elucidated. A protein kinase activity was identified as alarge multisubunit complex which can phosphorylate IκBα at Ser 32 and 36(Chen et al., 1996; Lee et al., 1997). Most recently, two relatedkinases have been cloned which contain a catalytic domain at theamino-terminus and a leucine zipper (LZ) as well as a helix-loop helix(HLH) motif at the carboxy terminus (Didonato et al., 1997; Mercurio etal., 1997; Regnier et al., 1997; Woronicz et al., 1997; Zandi et al.,1997). Although both of them have been shown to be essentialcontributors to cytokine-mediated NF-κB activation, understanding of theprecise nature of the IκB kinase activity and its regulatory mechanismsneeded further investigation and identification of the other subunits ofthe kinase complex. Another important issue unanswered was how discreteactivation signals triggered by a variety of known stimulators areintegrated to give rise to IκB kinase activity.

One attractive approach to such questions would be the use of somaticcell genetics. Although the diploidy of the mammalian genome presents amajor hurdle to a genetic approach, successful establishment ofrecessive mutants has provided helpful information on a signalingpathway and a reliable way to identify relevant gene(s) bycomplementation. Indeed, the Janus kinase family of tyrosine kinases wasidentified as essential signal transducers for the interferons through agenetic approach (Darnell et al., 1994; Velazquez et al., 1992).Concerning the NF-κB signaling pathways, the characterization of amutant of the murine pre-B cell line 70Z/3, 1.3E2, has previously beenreported, which had been isolated by selecting cells unable to expresssurface IgM following lipopolysaccharide stimulation (Courtois et al.,1997).

The recent description of a high molecular weight cytoplasmic complexable to phosphorylate IκBα on serines 32 and 36 (Chen et al., 1996; Leeet al., 1997) has prompted intense studies, which culminated a fewmonths ago with the cloning of two kinases, named IKK-1 and IKK-2, orIKKα and IKKβ (Didonato et al., 1997; Mercurio et al., 1997; Regnier etal., 1997; Woronicz et al., 1997; Zandi et al., 1997). Two approacheswere used to this end: one involved biochemical purification from acytoplasmic extract derived from TNF-treated HeLa cells (Didonato etal., 1997; Mercurio et al., 1997; Zandi et al., 1997), while the otherused a 2-hybrid screen using as a bait NIK, a protein kinase previouslyshown to be involved in TNF- and IL-1-induced NF-κB activation (Regnieret al., 1997; Woronicz et al., 1997). The cloned kinases were postulatedto directly phosphorylate serines 32 and 36 of IκBα, although this hasnot been formally demonstrated. The reason for this uncertainty is thatall kinase assays reported so far rely on immunoprecipitation oftransfected or in vitro translated IKK, therefore leaving open thepossibility that the “true” IκB kinase is coprecipitated together withIKK and the rest of the high molecular weight complex.Immunoprecipitation of one kinase from extracts of cells transfectedwith the two kinases results in the coprecipitation of the secondkinase, and a more detailed study has demonstrated thathetero-association was favored over homo-association. The sequence ofIKK-1 and IKK-2 has revealed two interesting features: a leucine zipperand a helix-loop-helix motif. Deletion of the LZ in one of the kinasesresults in the abrogation of coimmunoprecipitation with either itself orthe other kinase, and a strong reduction in the resulting kinaseactivity. However it is unclear whether the LZ motif is required fordirect interaction between the kinase subunits or between the kinase(s)and some other component of the complex. Deletion of the HLH motifleaves the coimmunoprecipitation of the two kinases intact, but stronglyreduces the resulting kinase activity. In the assays used in the abovementioned papers, transfected IKK-2 seems to exhibit a stronger basalkinase activity when compared to IKK-1 (Mercurio et al., 1997; Zandi etal., 1997). Zandi et al. (Zandi et al., 1997) also observed thatcotranslation of the two kinases in wheat germ extracts resulted in noIκB kinase activity, suggesting that either post-translationalmodifications or additional components of the complex (or both) arerequired. Cotranslation of the two kinases in wheat germ extractsprecluded their association. One possibility is that the kinase subunitsneed to be incorporated into the 600-800 kD complex in order to be fullyactive, and that some critical components of the complex are absent inwheat germ extracts. In any case all these data emphasize the importanceof identifying additional components of the complex.

If the identity of molecules involved in NF-κB activation were known,one could block NF-κB activation, and thereby treat cellulardysfunctions associated therewith, by inactivating these molecules.

In view of the aforementioned limited information regarding moleculesinvolved in NF-κB activation, it is clear that there exists a need inthe art for identifying the sequences encoding such molecules.

SUMMARY OF THE INVENTION

Accordingly, one object of this invention is to provide a modulator ofNF-κB and its subunits in purified form that exhibits certaincharacteristics and activities associated with inhibition of NF-κBactivity.

It is a further object of the present invention to provide antibodies tothe modulator of NF-κB and its subunits, and methods for theirpreparation, including recombinant means.

It is a further object of the present invention to provide a method fordetecting the presence of the modulator of NF-κB and its subunits inmammals in which invasive, spontaneous, or idiopathic pathologicalstates are suspected to be present.

It is a further object of the present invention to provide a method andassociated assay system for screening substances such as drugs, agentsand the like, potentially effective in either mimicking the activity orfighting against the adverse effects of the modulator of NF-κB and/orits subunits in mammals.

It is a still further object of the present invention to provide amethod for the treatment of mammals to control the transcriptionalactivity induced by NF-κB, so as to alter the adverse consequences ofsuch presence or activity, or where beneficial, to enhance suchactivity.

It is a still further object of the present invention to provide amethod for the treatment of mammals to control the amount or activity ofthe transcriptional activity of NF-κB, so as to treat or avert theadverse consequences of invasive, spontaneous or idiopathic pathologicalstates.

It is a still further object of the present invention to providepharmaceutical compositions for use in therapeutic methods whichcomprise or are based upon the modulator of NF-κB, its subunits, theirbinding partner(s), or upon agents or drugs that control the production,or that mimic or antagonize the activities of the modulator of NF-κB.

With the foregoing and other objects, advantages and features of theinvention that will become hereinafter apparent, the nature of theinvention may be more clearly understood by reference to the followingdetailed description of the preferred embodiments of the invention andto the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1. Characterisation of 5R cells. A. Fifty μg of whole cell extractsderived from wild-type Rat-1 cells (lane 1), the Tax-transformed cloneM319-5 (lane 2), the 5R flat revertant (lane 3) and a pool of hybridsbetween 5R and a Rat-1 derived clone bearing an integrated hygromycinresistance gene (lane 4) were analyzed by immunoblotting using anti TaxmAb M173.

B. Five μg of nuclear extracts derived from the same cells (as indicatedabove the lanes) were analyzed by bandshift assay using the κB sitederived from the H-2 K^(b) promoter as a probe. The NF-κB complex isindicated by a square dot on the right. C, D. Rat-1 or 5R cells werecotransfected with 0.25 μg of HTLV-1 LTR-luciferase (panel C) orIgκ-luciferase (panel D) and 1 μg of either empty vector (C) or Tax orrelA expression vectors. Luciferase activity was measured after 40hours. Fold induction over basal level is shown.E. Rat-1 or 5R cells (as indicated) were co-cultured with Rat-1 cellscarrying an integrated Igκ-luciferase plasmid, treated with (+) orwithout (−) 50% PEG for 1 minute and harvested 12 hours later.Equivalent amount of protein extract was used for the luciferase assay.

FIG. 2. Response of Rat-1 and 5R cells to NF-κB activating signals.

A. Bandshift assay of nuclear extracts from Rat-1 or 5R cells eitheruntreated (none) or stimulated as indicated above the lanes. Stimulationwas for 30 minutes with 10 ng/ml of TNF-α, 20 ng/ml of IL-1, 15 μg/ml ofLPS or 0.1 mg/ml of dsRNA.B. Transactivation of Igκ-luciferase transfected Rat-1 or 5R cells byTNF-α (T), IL-1 (I), LPS (L) or dsRNA (R). Stimulation was for 3 hours.C. Immunoblotting analysis of extracts derived from LPS treated Rat-1 or5R cells. Cytoplasmic extracts were prepared at the indicated times and50 μg analyzed by Western blotting.

FIG. 3. Sequence of the NEMO protein. The putative leucine zipper isboxed.

FIG. 4. NEMO complements the defect in 5R cells. A. Rat-1 or 5R cellswere transiently transfected with 0.25 μg of Igκ-luciferase and theindicated amount of CMV-hygro-NEMO. Luciferase assays were performed asdescribed in FIG. 2.

B. Band shift assay of Rat-1- or 5R-derived cell lines stably expressingNEMO. Five μg of nuclear extracts derived from the following cell lineswere analyzed as in FIG. 1. Lane 1: wild-type Rat-1 cells. Lane 2: apool of Rat-1 cells transfected with CMV-hygro-NEMO. Lane 3: 5R cells.Lane 4: h12 cells (5R cells containing the inducible blasticidin Sresistance gene). Lanes 5, 6: cDNA library infected h12 clones thatsurvived the blasticidin S selection. The size of the cDNA amplifiedfrom each clone is indicated. Lanes 7, 8: Independent pools of 5R cellsstably transfected with CMV-hygro-NEMO. Lanes 9, 10: two representative5R cell clones obtained by stable transfection with CMV-hygro-NEMO.C. Immunoblotting analysis of cytoplasmic extracts (100 μg) derived fromRat-1 or 5R cells was carried out with an antibody specific for NEMO.rNEMO: rat NEMO.

FIG. 5. NEMO complements the defect in 1.3E2 cells. A. 1.3E2, 1.3E2stably transfected with NEMO (1.3E2N) and 70Z/3 cells were transientlycotransfected with 3 μg of Igκ-luciferase and 6 μg of CMV-hygro-NEMO.After 24 hours, cells were splitted in two and left untreated (−) orstimulated (+) with 15 g/ml LPS. Luciferase assays were performed asdescribed in FIG. 2.

B. Bandshift assay of complemented 1.3E2 cells. 70Z/3 (lanes 1-4), 1.3E2(lanes 5-8) or a pool of 1.3E2 cells stably transfected withCMV-hygro-NEMO (1.3E2N, lanes 9-12) were left untreated (lanes 1, 5 9),or stimulated with 15 μg/ml LPS (lanes 2, 6, 10), 100 ng/ml PMA (lanes3, 7, 11) or 20 ng/ml IL-1 (lanes 4, 8, 12). Five μg of nuclear extractswere then analyzed by bandshift using the H-2 K^(b) derived κB site.C. Immunoblotting analysis of cytoplasmic extracts (100 μg) derived from70Z/3 or 1.3E2 cells was carried out with the NEMO antiserum. mNEMO:mouse NEMO.

FIG. 6. NEMO is associated with an inducible endogenous IκBα kinaseactivity. Rat-1 or 5R cells were treated for 5 minutes with or withoutTNF-α (10 ng/ml). Cytoplasmic extracts were immunoprecipitated witheither preimmune serum (P.I.), anti-IKK-1 antibody (anti-IKK-1) or NEMOantiserum (anti-NEMO) and specific IκBα kinase activity was determinedby an in vitro immune complex kinase assay with GST-IκBα (1-72) wildtype or GST-IκBα (1-72) S32A/S36A mutant protein as substrates.

FIG. 7. NEMO is a subunit of the IκB kinase complex

A. Gel filtration analysis of NEMO and IκB kinase complex in Rat-1 and5R cells. S100 extracts were prepared as described in Materials andMethods and fractionated through a Superose 6 column. Fractions wereanalyzed by Western blotting, using antibodies specific for IKK-1 orNEMO. Analysis of NF-κB/IκB elution, using an anti-relA antibody is alsoshown. To demonstrate identical elution of Rat-1 and 5R extracts, theprotein profile from each fraction was analyzed by silver staining(Upper pannel).B. Communoprecipitation of IKK-1 with NEMO. Positive NEMO fractions fromRat-1 and the equivalent fractions from 5R cells were immunoprecipitatedwith anti-NEMO, run through a 7.5% SDS-Laemmli gel and immunoblottedwith anti-IKK-1.C. NEMO forms homodimers. The NEMO protein was in vitro synthesized inwheat germ extract and treated with the indicated concentrations ofglutaraldehyde. The reactions were immunoprecipitated with NEMOantiserum and analysed on a 8% SDS-polyacrylamide gel. The positions ofthe NEMO monomer and NEMO dimer ((NEMO)₂) are indicated. Lo; in vitrotranslated product.D. In vitro interaction between NEMO and IKK-2. Untagged NEMO (lane 1),VSV-IKK 2 (lane 3), or both molecules (lane 2) were in vitro translatedin wheat germ extract (Load). The ³⁵S labelled products were thenprecipitated with anti-VSV antibody (VSV-IP). Lane 4 representsunprogrammed wheat germ extract. The relevant proteins are indicated onthe right.

FIG. 8. Nucleotide sequence of NEMO.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, “Molecular Cloning:A Laboratory Manual” (1989); “Current Protocols in Molecular Biology”Volumes I-III [Ausubel, R. M., ed. (1994)]; “Cell Biology: A LaboratoryHandbook” Volumes I-III [J. E. Celis, ed. (1994))]; “Current Protocolsin Immunology” Volumes I-III [Coligan, J. E., ed. (1994)];“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “TranscriptionAnd Translation” [B. D. Hames & S. J. Higgins, eds. (1984)]; “AnimalCell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells AndEnzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To MolecularCloning” (1984).

Therefore, if appearing herein, the following terms shall have thedefinitions set out below.

The term “modulator of NF-κB” and any variants not specifically listedas used throughout the present application and claims refer toproteinaceous material including single or multiple proteins, andextends to those proteins having the amino acid sequence data describedherein and presented in FIG. 3 (SEQ ID NO:1), and the profile ofactivities set forth herein and in the Claims. Accordingly, proteinsdisplaying substantially equivalent or altered activity are likewisecontemplated. These modifications may be deliberate, for example, suchas modifications obtained through site-directed mutagenesis, or may beaccidental, such as those obtained through mutations in hosts that areproducers of the complex or its named subunits. Also, the term“modulator of NF-κB” is intended to include within its scope proteinsspecifically recited herein as well as all substantially homologousanalogs and allelic variations.

The amino acid residues described herein are preferred to be in the “L”isomeric form. However, residues in the “D” isomeric form can besubstituted for any L-amino acid residue, as long as the desiredfunctional property of immunoglobulin-binding is retained by thepolypeptide. NH₂ refers to the free amino group present at the aminoterminus of a polypeptide. COOH refers to the free carboxy group presentat the carboxy terminus of a polypeptide.

It should be noted that all amino-acid residue sequences are representedherein by formulae whose left and right orientation is in theconventional direction of amino-terminus to carboxy-terminus.Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates a peptide bond to a furthersequence of one or more amino-acid residues.

A “replicon” is any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo; i.e.,capable of replication under its own control.

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment.

A “DNA molecule” refers to the polymeric form of deoxyribonucleotides(adenine, guanine, thymine, or cytosine) in its either single strandedform, or a double-stranded helix. This term refers only to the primaryand secondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (e.g., restrictionfragments), viruses, plasmids, and chromosomes. In discussing thestructure of particular double-stranded DNA molecules, sequences may bedescribed herein according to the normal convention of giving only thesequence in the 5′ to 3′ direction along the nontranscribed strand ofDNA (i.e., the strand having a sequence homologous to the mRNA).

An “origin of replication” refers to those DNA sequences thatparticipate in DNA synthesis.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in vivo when placed underthe control of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus. Acoding sequence can include, but is not limited to, prokaryoticsequences, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, polyadenylation signals,terminators, and the like, that provide for the expression of a codingsequence in a host cell.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined by mapping with nuclease S1), as well as protein binding domains(consensus sequences) responsible for the binding of RNA polymerase.Eukaryotic promoters will often, but not always, contain “TATA” boxesand “CAT” boxes. Prokaryotic promoters contain Shine-Dalgarno sequencesin addition to the −10 and −35 consensus sequences.

An “expression control sequence” is a DNA sequence that controls andregulates the transcription and translation of another DNA sequence. Acoding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence.

A “signal sequence” can be included before the coding sequence. Thissequence encodes a signal peptide, N-terminal to the polypeptide, thatcommunicates to the host cell to direct the polypeptide to the cellsurface or secrete the polypeptide into the media, and this signalpeptide is clipped off by the host cell before the protein leaves thecell. Signal sequences can be found associated with a variety ofproteins native to prokaryotes and eukaryotes.

The term “oligonucleotide,” as used herein in referring to the probe ofthe present invention, is defined as a molecule comprised of two or moreribonucleotides, preferably more than three. Its exact size will dependupon many factors which, in turn, depend upon the ultimate function anduse of the oligonucleotide.

The term “primer” as used herein refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product, which is complementary to a nucleic acid strand, isinduced, i.e., in the presence of nucleotides and an inducing agent suchas a DNA polymerase and at a suitable temperature and pH. The primer maybe either single-stranded or double-stranded and must be sufficientlylong to prime the synthesis of the desired extension product in thepresence of the inducing agent. The exact length of the primer willdepend upon many factors, including temperature, source of primer anduse of the method. For example, for diagnostic applications, dependingon the complexity of the target sequence, the oligonucleotide primertypically contains 15-25 or more nucleotides, although it may containfewer nucleotides.

The primers herein are selected to be “substantially” complementary todifferent strands of a particular target DNA sequence. This means thatthe primers must be sufficiently complementary to hybridize with theirrespective strands. Therefore, the primer sequence need not reflect theexact sequence of the template. For example, a non-complementarynucleotide fragment may be attached to the 5′ end of the primer, withthe remainder of the primer sequence being complementary to the strand.Alternatively, non-complementary bases or longer sequences can beinterspersed into the primer, provided that the primer sequence hassufficient complementarity with the sequence of the strand to hybridizetherewith and thereby form the template for the synthesis of theextension product.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

A cell has been “transformed” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. The transforming DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA. A “clone” is a population of cells derived from a single cell orcommon ancestor by mitosis. A “cell line” is a clone of a primary cellthat is capable of stable growth in vitro for many generations.

Two DNA sequences are “substantially homologous” when at least about 75%(preferably at least about 80%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra.

It should be appreciated that also within the scope of the presentinvention are DNA sequences encoding modulators of NF-κB which code fora modulator of NF-κB having the same amino acid sequence as SEQ ID NO:1,but which are degenerate to SEQ ID NO:2. By “degenerate to” is meantthat a different three-letter codon is used to specify a particularamino acid. It is well known in the art that the following codons can beused interchangeably to code for each specific amino acid:

Phenylalanine (Phe or F) UUU or UUC Leucine (Leu or L) UUA or UUG or CUUor CUC or CUA or CUG Isoleucine (Ile or I) AUU or AUC or AUA Methionine(Met or M) AUG Valine (Val or V) GUU or GUC of GUA or GUG Serine (Ser orS) UCU or UCC or UCA or UCG or AGU or AGC Proline (Pro or P) CCU or CCCor CCA or CCG Threonine (Thr or T) ACU or ACC or ACA or ACG Alanine (Alaor A) GCU or GCG or GCA or GCG Tyrosine (Tyr or Y) UAU or UAC Histidine(His or H) CAU or CAC Glutamine (Gln or Q) CAA or CAG Asparagine (Asn orN) AAU or AAC Lysine (Lys or K) AAA or AAG Aspartic Acid (Asp or D) GAUor GAC Glutamic Acid (Glu or E) GAA or GAG Cysteine (Cys or C) UGU orUGC Arginine (Arg or R) CGU or CGC or CGA or CGG or AGA or AGG Glycine(Gly or G) GGU or GGC or GGA or GGG Tryptophan (Trp or W) UGGTermination codon UAA (ochre) or UAG (amber) or UGA (opal)

It should be understood that the codons specified above are for RNAsequences. The corresponding codons for DNA have a T substituted for U.

Mutations can be made in SEQ ID NO:2 such that a particular codon ischanged to a codon which codes for a different amino acid. Such amutation is generally made by making the fewest nucleotide changespossible. A substitution mutation of this sort can be made to change anamino acid in the resulting protein in a non-conservative manner (i.e.,by changing the codon from an amino acid belonging to a grouping ofamino acids having a particular size or characteristic to an amino acidbelonging to another grouping) or in a conservative manner (i.e., bychanging the codon from an amino acid belonging to a grouping of aminoacids having a particular size or characteristic to an amino acidbelonging to the same grouping). Such a conservative change generallyleads to less change in the structure and function of the resultingprotein. A non-conservative change is more likely to alter thestructure, activity or function of the resulting protein. The presentinvention should be considered to include sequences containingconservative changes which do not significantly alter the activity orbinding characteristics of the resulting protein.

The following is one example of various groupings of amino acids:

Amino Acids with Nonpolar R Groups

Alanine Valine Leucine Isoleucine Proline Phenylalanine TryptophanMethionine

Amino Acids with Uncharged Polar R Groups

Glycine Serine Threonine Cysteine Tyrosine Asparagine Glutamine

Amino Acids with Charged Polar R Groups (Negatively Charged at Ph 6.0)Aspartic acidGlutamic acid

Basic Amino Acids (Positively Charged at pH 6.0) Lysine ArginineHistidine (at pH 6.0)

Another grouping may be those amino acids with phenyl groups:

Phenylalanine Tryptophan Tyrosine

Another grouping may be according to molecular weight (i.e., size of Rgroups):

Glycine  75 Alanine  89 Serine 105 Proline 115 Valine 117 Threonine 119Cysteine 121 Leucine 131 Isoleucine 131 Asparagine 132 Aspartic acid 133Glutamine 146 Lysine 146 Glutamic acid 147 Methionine 149 Histidine (atpH 6.0) 155 Phenylalanine 165 Arginine 174 Tyrosine 181 Tryptophan 204Particularly preferred substitutions are:

Lys for Arg and vice versa such that a positive charge may bemaintained;

Glu for Asp and vice versa such that a negative charge may bemaintained;

Ser for Thr such that a free —OH can be maintained; and

Gln for Asn such that a free NH₂ can be maintained.

Amino acid substitutions may also be introduced to substitute an aminoacid with a particularly preferable property. For example, a Cys may beintroduced a potential site for disulfide bridges with another Cys. AHis may be introduced as a particularly “catalytic” site (i.e., His canact as an acid or base and is the most common amino acid in biochemicalcatalysis). Pro may be introduced because of its particularly planarstructure, which induces β-turns in the protein's structure.

Two amino acid sequences are “substantially homologous” when at leastabout 70% of the amino acid residues (preferably at least about 80%, andmost preferably at least about 90 or 95%) are identical, or representconservative substitutions.

A “heterologous” region of the DNA construct is an identifiable segmentof DNA within a larger DNA molecule that is not found in associationwith the larger molecule in nature. Thus, when the heterologous regionencodes a mammalian gene, the gene will usually be flanked by DNA thatdoes not flank the mammalian genomic DNA in the genome of the sourceorganism. Another example of a heterologous coding sequence is aconstruct where the coding sequence itself is not found in nature (e.g.,a cDNA where the genomic coding sequence contains introns, or syntheticsequences having codons different than the native gene). Allelicvariations or naturally-occurring mutational events do not give rise toa heterologous region of DNA as defined herein.

An “antibody” is any immunoglobulin, including antibodies and fragmentsthereof, that binds a specific epitope. The term encompasses polyclonal,monoclonal, and chimeric antibodies, the last mentioned described infurther detail in U.S. Pat. Nos. 4,816,397 and 4,816,567.

An “antibody combining site” is that structural portion of an antibodymolecule comprised of heavy and light chain variable and hypervariableregions that specifically binds antigen.

The phrase “antibody molecule” in its various grammatical forms as usedherein contemplates both an intact immunoglobulin molecule and animmunologically active portion of an immunoglobulin molecule.

Exemplary antibody molecules are intact immunoglobulin molecules,substantially intact immunoglobulin molecules and those portions of animmunoglobulin molecule that contains the paratope, including thoseportions known in the art as Fab, Fab′, F(ab′)₂ and F(v), which portionsare preferred for use in the therapeutic methods described herein.

Fab and F(ab′)₂ portions of antibody molecules are prepared by theproteolytic reaction of papain and pepsin, respectively, onsubstantially intact antibody molecules by methods that are well-known.See for example, U.S. Pat. No. 4,342,566 to Theofilopolous et al. Fab′antibody molecule portions are also well-known and are produced fromF(ab′)₂ portions followed by reduction of the disulfide bonds linkingthe two heavy chain portions as with mercaptoethanol, and followed byalkylation of the resulting protein mercaptan with a reagent such asiodoacetamide. An antibody containing intact antibody molecules ispreferred herein.

The phrase “monoclonal antibody” in its various grammatical forms refersto an antibody having only one species of antibody combining sitecapable of immunoreacting with a particular antigen. A monoclonalantibody thus typically displays a single binding affinity for anyantigen with which it immunoreacts. A monoclonal antibody may thereforecontain an antibody molecule having a plurality of antibody combiningsites, each immunospecific for a different antigen; e.g., a bispecific(chimeric) monoclonal antibody.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human.

The phrase “therapeutically effective amount” is used herein to mean anamount sufficient to prevent, and preferably reduce by at least about 30percent, more preferably by at least 50 percent, most preferably by atleast 90 percent, a clinically significant change in the S phaseactivity of a target cellular mass, or other feature of pathology suchas for example, elevated blood pressure, fever or white cell count asmay attend its presence and activity.

A DNA sequence is “operatively linked” to an expression control sequencewhen the expression control sequence controls and regulates thetranscription and translation of that DNA sequence. The term“operatively linked” includes having an appropriate start signal (e.g.,ATG) in front of the DNA sequence to be expressed and maintaining thecorrect reading frame to permit expression of the DNA sequence under thecontrol of the expression control sequence and production of the desiredproduct encoded by the DNA sequence. If a gene that one desires toinsert into a recombinant DNA molecule does not contain an appropriatestart signal, such a start signal can be inserted in front of the gene.

The term “standard hybridization conditions” refers to salt andtemperature conditions substantially equivalent to 5×SSC and 65° C. forboth hybridization and wash. However, one skilled in the art willappreciate that such “standard hybridization conditions” are dependenton particular conditions including the concentration of sodium andmagnesium in the buffer, nucleotide sequence length and concentration,percent mismatch, percent formamide, and the like. Also important in thedetermination of “standard hybridization conditions” is whether the twosequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such standardhybridization conditions are easily determined by one skilled in the artaccording to well known formulae, wherein hybridization is typically10-20° C. below the predicted or determined T_(m) with washes of higherstringency, if desired.

In its primary aspect, the present invention concerns the identificationof a modulator of NF-κB.

In a particular embodiment, the present invention relates to a modulatorof NF-κB termed NF-κB Essential Modulator (NEMO)

As stated above, the present invention also relates to a recombinant DNAmolecule or cloned gene, or a degenerate variant thereof, which encodesa modulator of NF-κB, or a fragment thereof, that possesses a molecularweight of about 48 kD and an amino acid sequence set forth in FIG. 3(SEQ ID NO:1); preferably a nucleic acid molecule, in particular arecombinant DNA molecule or cloned gene, encoding the 48 kD modulator ofNF-κB has a nucleotide sequence or is complementary to a DNA sequenceshown in FIG. 8 (SEQ ID NO:2).

The possibilities both diagnostic and therapeutic that are raised by theexistence of the modulator of NF-κB, derive from the fact that thismodulator appears to participate in direct and causal protein-proteininteraction with at least one of the IκB kinases, which is involved inthe activation of NF-κB. As suggested earlier and elaborated further onherein, the present invention contemplates pharmaceutical interventionin the cascade of reactions in which the modulator of NF-κB isimplicated, to modulate the activity initiated by the modulator ofNF-κB.

Thus, in instances where it is desired to reduce or inhibit themodulator of NF-κB resulting from a particular stimulus or factor, anappropriate inhibitor of the modulator of NF-κB could be introduced toblock the interaction of the modulator of NF-κB with those factors towhich the modulator of NF-κB binds (e.g., the IκB kinase).Correspondingly, instances where insufficient NF-κB-inducedtranscriptional activity is taking place could be remedied by theintroduction of additional quantities of the modulator of NF-κB or itschemical or pharmaceutical cognates, analogs, fragments and the like.

As discussed earlier, the modulator of NF-κB or its binding partners orother ligands or agents exhibiting either mimicry or antagonism to themodulator of NF-κB or control over its production, may be prepared inpharmaceutical compositions, with a suitable carrier and at a strengtheffective for administration by various means to a patient experiencingan adverse medical condition associated with NF-κB activation for thetreatment thereof. A variety of administrative techniques may beutilized, among them parenteral techniques such as subcutaneous,intravenous and intraperitoneal injections, catheterizations and thelike. Average quantities of the modulator of NF-κB or its subunits mayvary and in particular should be based upon the recommendation andprescription of a qualified physician or veterinarian.

Also, antibodies including both polyclonal and monoclonal antibodies,and drugs that modulate the production or activity of the modulator ofNF-κB and/or its subunits may possess certain diagnostic applicationsand may for example, be utilized for the purpose of detecting and/ormeasuring conditions such as viral infection or the like. For example,the modulator of NF-κB or its subunits may be used to produce bothpolyclonal and monoclonal antibodies to themselves in a variety ofcellular media, by known techniques such as the hybridoma techniqueutilizing, for example, fused mouse spleen lymphocytes and myelomacells. Likewise, small molecules that mimic or antagonize theactivity(ies) of the modulator of NF-κB of the invention may bediscovered or synthesized, and may be used in diagnostic and/ortherapeutic protocols.

The general methodology for making monoclonal antibodies by hybridomasis well known. Immortal, antibody-producing cell lines can also becreated by techniques other than fusion, such as direct transformationof B lymphocytes with oncogenic DNA, or transfection with Epstein-Barrvirus. See, e.g., M. Schreier et al., “Hybridoma Techniques” (1980);Hammerling et al., “Monoclonal Antibodies And T-cell Hybridomas” (1981);Kennett et al., “Monoclonal Antibodies” (1980); see also U.S. Pat. Nos.4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917;4,472,500; 4,491,632; 4,493,890.

Panels of monoclonal antibodies produced against modulator of NF-κBpeptides can be screened for various properties; i.e., isotype, epitope,affinity, etc. Of particular interest are monoclonal antibodies thatneutralize the activity of the modulator of NF-κB or its subunits. Highaffinity antibodies are also useful when immunoaffinity purification ofnative or recombinant modulator of NF-κB is possible.

Preferably, the anti-NF-κB modulator antibody used in the diagnosticmethods of this invention is an affinity purified polyclonal antibody.More preferably, the antibody is a monoclonal antibody (mAb). Inaddition, it is preferable for the anti-NF-κB modulator antibodymolecules used herein be in the form of Fab, Fab′, F(ab′)₂ or F(v)portions of whole antibody molecules.

Methods for producing polyclonal anti-polypeptide antibodies arewell-known in the art. See U.S. Pat. No. 4,493,795 to Nestor et al. Amonoclonal antibody, typically containing Fab and/or F(ab′)₂ portions ofuseful antibody molecules, can be prepared using the hybridomatechnology described in Antibodies—A Laboratory Manual, Harlow and Lane,eds., Cold Spring Harbor Laboratory, New York (1988), which isincorporated herein by reference. Briefly, to form the hybridoma fromwhich the monoclonal antibody composition is produced, a myeloma orother self-perpetuating cell line is fused with lymphocytes obtainedfrom the spleen of a mammal hyperimmunized with a NF-κBmodulator-binding portion thereof, or NF-κB modulator.

Splenocytes are typically fused with myeloma cells using polyethyleneglycol (PEG) 6000. Fused hybrids are selected by their sensitivity toHAT. Hybridomas producing a monoclonal antibody useful in practicingthis invention are identified by their ability to immunoreact with thepresent NF-κB modulator.

A monoclonal antibody useful in practicing the present invention can beproduced by initiating a monoclonal hybridoma culture comprising anutrient medium containing a hybridoma that secretes antibody moleculesof the appropriate antigen specificity. The culture is maintained underconditions and for a time period sufficient for the hybridoma to secretethe antibody molecules into the medium. The antibody-containing mediumis then collected. The antibody molecules can then be further isolatedby well-known techniques.

Media useful for the preparation of these compositions are bothwell-known in the art and commercially available and include syntheticculture media, inbred mice and the like. An exemplary synthetic mediumis Dulbecco's minimal essential medium (DMEM; Dulbecco et al., Virol.8:396 (1959)) supplemented with 4.5 gm/l glucose, 20 mm glutamine, and20% fetal calf serum. An exemplary inbred mouse strain is the Balb/c.

Methods for producing monoclonal anti-NF-κB modulator antibodies arealso well-known in the art. See Niman et al., Proc. Natl. Acad. Sci.USA, 80:4949-4953 (1983). Typically, the present NF-κB modulator or apeptide analog is used either alone or conjugated to an immunogeniccarrier, as the immunogen in the before described procedure forproducing anti-NF-κB modulator monoclonal antibodies. The hybridomas arescreened for the ability to produce an antibody that immunoreacts withthe NF-κB modulator peptide analog and the present NF-κB modulator.

The present invention further contemplates therapeutic compositionsuseful in practicing the therapeutic methods of this invention. Asubject therapeutic composition includes, in admixture, apharmaceutically acceptable excipient (carrier) and one or more of aNF-κB modulator, polypeptide analog thereof or fragment thereof, asdescribed herein as an active ingredient. In a preferred embodiment, thecomposition comprises an antigen capable of modulating the specificbinding of the present NF-κB modulator within a target cell.

The preparation of therapeutic compositions which contain polypeptides,analogs or active fragments as active ingredients is well understood inthe art. Typically, such compositions are prepared as injectables,either as liquid solutions or suspensions, however, solid forms suitablefor solution in, or suspension in, liquid prior to injection can also beprepared. The preparation can also be emulsified. The active therapeuticingredient is often mixed with excipients which are pharmaceuticallyacceptable and compatible with the active ingredient. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanol,or the like and combinations thereof. In addition, if desired, thecomposition can contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents which enhance theeffectiveness of the active ingredient.

A polypeptide, analog or active fragment can be formulated into thetherapeutic composition as neutralized pharmaceutically acceptable saltforms. Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide or antibodymolecule) and which are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like. Salts formed from thefree carboxyl groups can also be derived from inorganic bases such as,for example, sodium, potassium, ammonium, calcium, or ferric hydroxides,and such organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

The therapeutic polypeptide-, analog- or active fragment-containingcompositions are conventionally administered intravenously, as byinjection of a unit dose, for example. The term “unit dose” when used inreference to a therapeutic composition of the present invention refersto physically discrete units suitable as unitary dosage for humans, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with therequired diluent; i.e., carrier, or vehicle.

The present invention also relates to a recombinant DNA molecule orcloned gene, or a degenerate variant thereof, which encodes a NF-κBmodulator; preferably a nucleic acid molecule, in particular arecombinant DNA molecule or cloned gene, encoding the NF-κB modulatorhas a nucleotide sequence or is complementary to a DNA sequence shown inFIG. 8 (SEQ ID NO:2).

The human and murine DNA sequences of the NF-κB modulator of the presentinvention or portions thereof, may be prepared as probes to screen forcomplementary sequences and genomic clones in the same or alternatespecies. The present invention extends to probes so prepared that may beprovided for screening cDNA and genomic libraries for the NF-κBmodulator. For example, the probes may be prepared with a variety ofknown vectors, such as the phage λ vector. The present invention alsoincludes the preparation of plasmids including such vectors, and the useof the DNA sequences to construct vectors expressing antisense RNA orribozymes which would attack the mRNAs of the DNA sequence set forth inFIG. 8 (SEQ ID NO:2). Correspondingly, the preparation of antisense RNAand ribozymes are included herein.

The present invention also includes NF-κB modulator proteins having theactivities noted herein, and that display the amino acid sequences setforth and described above and having SEQ ID NO:1.

In a further embodiment of the invention, the full DNA sequence of therecombinant DNA molecule or cloned gene so determined may be operativelylinked to an expression control sequence which may be introduced into anappropriate host. The invention accordingly extends to unicellular hoststransformed with the cloned gene or recombinant DNA molecule comprisinga DNA sequence encoding the present NF-κB modulator(s), and moreparticularly, the complete DNA sequence determined from the sequencesset forth above and in SEQ ID NO:2.

According to other preferred features of certain preferred embodimentsof the present invention, a recombinant expression system is provided toproduce biologically active animal or human NF-κB modulator.

The concept of the NF-κB modulator contemplates that specific factorsexist for correspondingly specific ligands, such as the IκB kinase andthe like, as described earlier. Accordingly, the exact structure of eachNF-κB modulator will understandably vary so as to achieve this ligandand activity specificity. It is this specificity and the directinvolvement of the NF-κB modulator in the chain of events leading toNF-κB-induced transcriptional activity, that offers the promise of abroad spectrum of diagnostic and therapeutic utilities.

The present invention naturally contemplates several means forpreparation of the NF-κB modulator, including as illustrated hereinknown recombinant techniques, and the invention is accordingly intendedto cover such synthetic preparations within its scope. The isolation ofthe cDNA and amino acid sequences disclosed herein facilitates thereproduction of the NF-κB modulator by such recombinant techniques, andaccordingly, the invention extends to expression vectors prepared fromthe disclosed DNA sequences for expression in host systems byrecombinant DNA techniques, and to the resulting transformed hosts.

The invention includes an assay system for screening of potential drugseffective to modulate NF-κB modulator activity of target mammalian cellsby interrupting or potentiating the nuclear translocation of NF-κB. Inone instance, the test drug could be administered to a cellular sample,to determine its effect upon the binding activity of the NF-κB modulatorto any chemical sample (including the IκB kinase), or to the test drug,by comparison with a control.

The assay system could more importantly be adapted to identify drugs orother entities that are capable of binding to the NF-κB modulator,thereby inhibiting or potentiating NF-κB-induced transcriptionalactivity. Such assay would be useful in the development of drugs thatwould be specific against particular cellular activity, or that wouldpotentiate such activity, in time or in level of activity. For example,such drugs might be used to modulate immune responses, stress responses,inflammation or apoptosis, or to treat other pathologies, as forexample, viral infection.

In yet a further embodiment, the invention contemplates antagonists ofthe activity of a NF-κB modulator.

The present invention likewise extends to the development of antibodiesagainst the NF-κB modulator(s), including naturally raised andrecombinantly prepared antibodies. For example, the antibodies could beused to screen expression libraries to obtain the gene or genes thatencode the NF-κB modulator(s). Such antibodies could include bothpolyclonal and monoclonal antibodies prepared by known genetictechniques, as well as bi-specific (chimeric) antibodies, and antibodiesincluding other functionalities suiting them for additional diagnosticuse conjunctive with their capability of modulating NF-κB activity Thus,the NF-κB modulator, its analogs, and any antagonists or antibodies thatmay be raised thereto, are capable of use in connection with variousdiagnostic techniques, including immunoassays, such as aradioimmunoassay, using for example, an antibody to the NF-κB modulatorthat has been labeled by either radioactive addition, orradioiodination.

In an immunoassay, a control quantity of the antagonists or antibodiesthereto, or the like may be prepared and labeled with an enzyme, aspecific binding partner and/or a radioactive element, and may then beintroduced into a cellular sample. After the labeled material or itsbinding partner(s) has had an opportunity to react with sites within thesample, the resulting mass may be examined by known techniques, whichmay vary with the nature of the label attached.

In the instance where a radioactive label, such as the isotopes ³H, ¹⁴C,³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re areused, known currently available counting procedures may be utilized. Inthe instance where the label is an enzyme, detection may be accomplishedby any of the presently utilized calorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques known inthe art.

The present invention includes an assay system which may be prepared inthe form of a test kit for the quantitative analysis of the extent ofthe presence of the NF-κB modulator, or to identify drugs or otheragents that may mimic or block its activity. The system or test kit maycomprise a labeled component prepared by one of the radioactive and/orenzymatic techniques discussed herein, coupling a label to the NF-κBmodulator, its agonists and/or antagonists, and one or more additionalimmunochemical reagents, at least one of which is a free or immobilizedligand, capable either of binding with the labeled component, itsbinding partner, one of the components to be determined or their bindingpartner(s).

In a further embodiment, the present invention relates to certaintherapeutic methods which would be based upon the activity of the NF-κBmodulator(s), its subunits, or active fragments thereof, or upon agentsor other drugs determined to possess the same activity. A firsttherapeutic method is associated with the prevention of themanifestations of conditions causally related to or following from thenuclear translocation of NF-κB, and comprises administering an agentcapable of modulating the production and/or activity of the NF-κBmodulator or subunits thereof, either individually or in mixture witheach other in an amount effective to prevent the development of thoseconditions in the host. For example, drugs or other binding partners tothe NF-κB modulator or proteins may be administered to inhibit orpotentiate NF-κB activity.

More specifically, the therapeutic method generally referred to hereincould include the method for the treatment of various pathologies orother cellular dysfunctions and derangements by the administration ofpharmaceutical compositions that may comprise effective inhibitors orenhancers of activation of the NF-κB modulator or its subunits, or otherequally effective drugs developed for instance by a drug screening assayprepared and used in accordance with a further aspect of the presentinvention. For example, drugs or other binding partners to the NF-κBmodulator, as represented by SEQ ID NO:1, may be administered to inhibitor potentiate NF-κB-induced transcriptional activity.

Another feature of this invention is the expression of the DNA sequencesdisclosed herein. As is well known in the art, DNA sequences may beexpressed by operatively linking them to an expression control sequencein an appropriate expression vector and employing that expression vectorto transform an appropriate unicellular host.

Such operative linking of a DNA sequence of this invention to anexpression control sequence, of course, includes, if not already part ofthe DNA sequence, the provision of an initiation codon, ATG, in thecorrect reading frame upstream of the DNA sequence.

A wide variety of host/expression vector combinations may be employed inexpressing the DNA sequences of this invention. Useful expressionvectors, for example, may consist of segments of chromosomal,non-chromosomal and synthetic DNA sequences.

Suitable vectors include derivatives of SV40 and known bacterialplasmids, e.g., E. coli plasmids col E1, pCR1, pBR322, pMB9 and theirderivatives, plasmids such as RP4; phage DNAS, e.g., the numerousderivatives of phage λ, e.g., NM989, and other phage DNA, e.g., M13 andfilamentous single stranded phage DNA; yeast plasmids such as the 2μplasmid or derivatives thereof; vectors useful in eukaryotic cells, suchas vectors useful in insect or mammalian cells; vectors derived fromcombinations of plasmids and phage DNAs, such as plasmids that have beenmodified to employ phage DNA or other expression control sequences; andthe like.

Any of a wide variety of expression control sequences—sequences thatcontrol the expression of a DNA sequence operatively linked to it—may beused in these vectors to express the DNA sequences of this invention.Such useful expression control sequences include, for example, the earlyor late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lacsystem, the trp system, the TAC system, the TRC system, the LTR system,the major operator and promoter regions of phage λ, the control regionsof fd coat protein, the promoter for 3-phosphoglycerate kinase or otherglycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), thepromoters of the yeast α-mating factors, and other sequences known tocontrol the expression of genes of prokaryotic or eukaryotic cells ortheir viruses, and various combinations thereof.

A wide variety of unicellular host cells are also useful in expressingthe DNA sequences of this invention. These hosts may include well knowneukaryotic and prokaryotic hosts, such as strains of E. coli,Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animalcells, such as CHO, R1.1, B-W and L-M cells, African Green Monkey kidneycells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g.,Sf9), and human cells and plant cells in tissue culture.

It will be understood that not all vectors, expression control sequencesand hosts will function equally well to express the DNA sequences ofthis invention. Neither will all hosts function equally well with thesame expression system. However, one skilled in the art will be able toselect the proper vectors, expression control sequences, and hostswithout undue experimentation to accomplish the desired expressionwithout departing from the scope of this invention. For example, inselecting a vector, the host must be considered because the vector mustfunction in it. The vector's copy number, the ability to control thatcopy number, and the expression of any other proteins encoded by thevector, such as antibiotic markers, will also be considered.

In selecting an expression control sequence, a variety of factors willnormally be considered. These include, for example, the relativestrength of the system, its controllability, and its compatibility withthe particular DNA sequence or gene to be expressed, particularly asregards potential secondary structures. Suitable unicellular hosts willbe selected by consideration of, e.g., their compatibility with thechosen vector, their secretion characteristics, their ability to foldproteins correctly, and their fermentation requirements, as well as thetoxicity to the host of the product encoded by the DNA sequences to beexpressed, and the ease of purification of the expression products.

Considering these and other factors a person skilled in the art will beable to construct a variety of vector/expression control sequence/hostcombinations that will express the DNA sequences of this invention onfermentation or in large scale animal culture.

It is further intended that NF-κB modulator analogs may be prepared fromnucleotide sequences of the protein complex/subunit derived within thescope of the present invention. Analogs, such as fragments, may beproduced, for example, by pepsin digestion of NF-κB modulator material.Other analogs, such as muteins, can be produced by standardsite-directed mutagenesis of NF-κB modulator coding sequences. Analogsexhibiting “NF-κB modulator activity” such as small molecules, whetherfunctioning as promoters or inhibitors, may be identified by known invivo and/or in vitro assays.

As mentioned above, a DNA sequence encoding NF-κB modulator can beprepared synthetically rather than cloned. The DNA sequence can bedesigned with the appropriate codons for the NF-κB modulator amino acidsequence. In general, one will select preferred codons for the intendedhost if the sequence will be used for expression. The complete sequenceis assembled from overlapping oligonucleotides prepared by standardmethods and assembled into a complete coding sequence. See, e.g., Edge,Nature, 292:756 (1981); Nambair et al., Science, 223:1299 (1984); Jay etal., J. Biol. Chem., 259:6311 (1984).

Synthetic DNA sequences allow convenient construction of genes whichwill express NF-κB modulator analogs or “muteins”. Alternatively, DNAencoding muteins can be made by site-directed mutagenesis of nativeNF-κB modulator genes or cDNAs, and muteins can be made directly usingconventional polypeptide synthesis.

A general method for site-specific incorporation of unnatural aminoacids into proteins is described in Christopher J. Noren, Spencer J.Anthony-Cahill, Michael C. Griffith, Peter G. Schultz, Science,244:182-188 (April 1989). This method may be used to create analogs withunnatural amino acids.

The present invention extends to the preparation of antisenseoligonucleotides and ribozymes that may be used to interfere with theexpression of the NF-κB modulator at the translational level. Thisapproach utilizes antisense nucleic acid and ribozymes to blocktranslation of a specific mRNA, either by masking that mRNA with anantisense nucleic acid or cleaving it with a ribozyme.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule. (See Weintraub, 1990;Marcus-Sekura, 1988.) In the cell, they hybridize to that mRNA, forminga double stranded molecule. The cell does not translate an mRNA in thisdouble-stranded form. Therefore, antisense nucleic acids interfere withthe expression of mRNA into protein. Oligomers of about fifteennucleotides and molecules that hybridize to the AUG initiation codonwill be particularly efficient, since they are easy to synthesize andare likely to pose fewer problems than larger molecules when introducingthem into NF-κB modulator-producing cells. Antisense methods have beenused to inhibit the expression of many genes in vitro (Marcus-Sekura,1988; Hambor et al., 1988).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single stranded RNA molecules in a manner somewhatanalogous to DNA restriction endonucleases. Ribozymes were discoveredfrom the observation that certain mRNAs have the ability to excise theirown introns. By modifying the nucleotide sequence of these RNAs,researchers have been able to engineer molecules that recognize specificnucleotide sequences in an RNA molecule and cleave it (Cech, 1988.).Because they are sequence-specific, only mRNAs with particular sequencesare inactivated.

Investigators have identified two types of ribozymes, Tetrahymena-typeand “hammerhead”-type. (Hasselhoff and Gerlach, 1988) Tetrahymena-typeribozymes recognize four-base sequences, while “hammerhead”-typerecognize eleven- to eighteen-base sequences. The longer the recognitionsequence, the more likely it is to occur exclusively in the target mRNAspecies. Therefore, hammerhead-type ribozymes are preferable toTetrahymena-type ribozymes for inactivating a specific mRNA species, andeighteen base recognition sequences are preferable to shorterrecognition sequences.

The DNA sequences described herein may thus be used to prepare antisensemolecules against, and ribozymes that cleave mRNAs for NF-κB modulatorand its ligands.

The present invention also relates to a variety of diagnosticapplications, including methods for detecting the presence of stimulisuch as the earlier referenced polypeptide ligands, by reference totheir ability to elicit the activities which are mediated by the presentNF-κB modulator. As mentioned earlier, the NF-κB modulator can be usedto produce antibodies to itself by a variety of known techniques, andsuch antibodies could then be isolated and utilized as in tests for thepresence of particular NF-κB modulator activity in suspect target cells.

As described in detail above, antibody(ies) to the NF-κB modulator canbe produced and isolated by standard methods including the well knownhybridoma techniques. For convenience, the antibody(ies) to the NF-κBmodulator will be referred to herein as Ab₁ and antibody(ies) raised inanother species as Ab₂.

The presence of NF-κB modulator in cells can be ascertained by the usualimmunological procedures applicable to such determinations. A number ofuseful procedures are known. Three such procedures which are especiallyuseful utilize either the NF-κB modulator labeled with a detectablelabel, antibody Ab₁ labeled with a detectable label, or antibody Ab₂labeled with a detectable label. The procedures may be summarized by thefollowing equations wherein the asterisk indicates that the particle islabeled, and “NF-κBM” stands for the NF-κB modulator:

NF-κBM*+Ab ₁ =NF-κBM*Ab ₁  A.

NF-κBM+Ab*=NF-κBMAb ₁*  B.

NF-κBM+Ab ₁ +Ab ₂ *=NF-κBMAb ₁ Ab ₂*  C.

The procedures and their application are all familiar to those skilledin the art and accordingly may be utilized within the scope of thepresent invention. The “competitive” procedure, Procedure A, isdescribed in U.S. Pat. Nos. 3,654,090 and 3,850,752. Procedure C, the“sandwich” procedure, is described in U.S. Patent Nos. RE 31,006 and4,016,043. Still other procedures are known such as the “doubleantibody,” or “DASP” procedure.

In each instance, the NF-κB modulator forms complexes with one or moreantibody(ies) or binding partners and one member of the complex islabeled with a detectable label. The fact that a complex has formed and,if desired, the amount thereof, can be determined by known methodsapplicable to the detection of labels.

It will be seen from the above, that a characteristic property of Ab₂ isthat it will react with Ab₁. This is because Ab₁ raised in one mammalianspecies has been used in another species as an antigen to raise theantibody Ab₂. For example, Ab₂ may be raised in goats using rabbitantibodies as antigens. Ab₂ therefore would be anti-rabbit antibodyraised in goats. For purposes of this description and claims, Ab₁ willbe referred to as a primary or anti-NF-κB modulator antibody, and Ab₂will be referred to as a secondary or anti-Ab₁ antibody.

The labels most commonly employed for these studies are radioactiveelements, enzymes, chemicals which fluoresce when exposed to ultravioletlight, and others.

A number of fluorescent materials are known and can be utilized aslabels. These include, for example, fluorescein, rhodamine, auramine,Texas Red, AMCA blue and Lucifer Yellow. A particular detecting materialis anti-rabbit antibody prepared in goats and conjugated withfluorescein through an isothiocyanate.

The NF-κB modulator or its binding partner(s) can also be labeled with aradioactive element or with an enzyme. The radioactive label can bedetected by any of the currently available counting procedures. Thepreferred isotope may be selected from ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr,⁵⁷Co, ⁵⁸Co, ⁵⁹Te, ⁹⁰Y, ¹³¹I, and ¹⁸⁶Re.

Enzyme labels are likewise useful, and can be detected by any of thepresently utilized colorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques. Theenzyme is conjugated to the selected particle by reaction with bridgingmolecules such as carbodiimides, diisocyanates, glutaraldehyde and thelike. Many enzymes which can be used in these procedures are known andcan be utilized. The preferred are peroxidase, β-glucuronidase,β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plusperoxidase and alkaline phosphatase. U.S. Pat. Nos. 3,654,090;3,850,752; and 4,016,043 are referred to by way of example for theirdisclosure of alternate labeling material and methods.

In a further embodiment of this invention, commercial test kits suitablefor use by a medical specialist may be prepared to determine thepresence or absence of predetermined NF-κB modulator activity orpredetermined NF-κB modulator activity capability in suspected targetcells. In accordance with the testing techniques discussed above, oneclass of such kits will contain at least the labeled NF-κB modulator orits binding partner, for instance an antibody specific thereto, anddirections, of course, depending upon the method selected, e.g.,“competitive,” “sandwich,” “DASP” and the like. The kits may alsocontain peripheral reagents such as buffers, stabilizers, etc.

Accordingly, a test kit may be prepared for the demonstration of thepresence or capability of cells for predetermined NF-κB modulatoractivity, comprising:

(a) a predetermined amount of at least one labeled immunochemicallyreactive component obtained by the direct or indirect attachment of thepresent NF-κB modulator or a specific binding partner thereto, to adetectable label;

(b) other reagents; and

(c) directions for use of said kit.

More specifically, the diagnostic test kit may comprise:

(a) a known amount of the NF-κB modulator as described above (or abinding partner) generally bound to a solid phase to form animmunosorbent, or in the alternative, bound to a suitable tag, or pluralsuch end products, etc. (or their binding partners) one of each;

(b) if necessary, other reagents; and

(c) directions for use of said test kit.

In a further variation, the test kit may be prepared and used for thepurposes stated above, which operates according to a predeterminedprotocol (e.g. “competitive,” “sandwich,” “double antibody,” etc.), andcomprises:

(a) a labeled component which has been obtained by coupling the NF-κBmodulator to a detectable label;

(b) one or more additional immunochemical reagents of which at least onereagent is a ligand or an immobilized ligand, which ligand is selectedfrom the group consisting of:

-   -   (i) a ligand capable of binding with the labeled component (a);    -   (ii) a ligand capable of binding with a binding partner of the        labeled component (a);    -   (iii) a ligand capable of binding with at least one of the        component(s) to be determined; and    -   (iv) a ligand capable of binding with at least one of the        binding partners of at least one of the component(s) to be        determined; and

(c) directions for the performance of a protocol for the detectionand/or determination of one or more components of an immunochemicalreaction between the NF-κB modulator and a specific binding partnerthereto.

In accordance with the above, an assay system for screening potentialdrugs effective to modulate the activity of the NF-κB modulator may beprepared. The NF-κB modulator may be introduced into a test system, andthe prospective drug may also be introduced into the resulting cellculture, and the culture thereafter examined to observe any changes inthe NF-κB modulator activity of the cells, due either to the addition ofthe prospective drug alone, or due to the effect of added quantities ofthe known NF-κB modulator.

In accordance with the present invention, a mutant cell line, 5R, wasoriginally isolated as a cellular flat variant of Rat-1 fibroblaststransformed by the Tax protein of human T-cell leukemia virus type 1(HTLV-1). Tax is known to activate transcription from the HTLV-1 longterminal repeat, to cause permanent activation of many cellulartranscription factors including NF-κB and to give rise to cellulartransformation (for a review, see Yoshida et al., 1995). 5R cells carrya recessive cellular mutation which abolishes Tax-induced constitutiveNF-κB activity, therefore providing a potential mean of identifying acritical molecule involved in Tax-mediated NF-κB activation.Interestingly, 5R cells were found to be resistant to multiple NF-κBactivating stimuli besides Tax, suggesting they carried a mutation at aconverging regulatory step. 5R cells were used for a geneticcomplementation approach for the following reasons. First, as the screenwas based on the NF-κB-dependent expression of a drug resistance gene,the presence of Tax would ensure restoration of a permanent high NF-κBactivity following complementation. Second, Rat-1-derived cells growwell in the presence of a high NF-κB activity. Third, 5R cells areexpected to show a transformed phenotype following complementation. Herethe genetic complementation of 5R cells is described. By infection witha cDNA expression library cloned into a retroviral vector, complementedclones derived from 5R cells were obtained and expression of the clonedgene, nemo, also complements the defect in the 1.3E2 cell line and showthat NEMO is part of the high molecular weight IKK complex and isrequired for its formation.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

EXAMPLES Example 1 Materials and Methods

Cells and transfections. The 70Z/3 murine pre-B cell line and the NF-1unresponsive mutant 1.3E2 were maintained in RPMI medium supplementedwith 10% foetal calf serum and 50 μM β-mercaptoethanol. 70Z/3 and 1.3E2cells were transiently transfected as described (Courtois et al., 1997).Isolated stable clones were prepared as described (Whiteside et al.,1995). Rat-1 and 5R cells were grown in DMEM supplemented with 10%foetal calf serum and transfected using the calcium phosphatecoprecipitation method. For measurement of luciferase activity intransiently transfected Rat-1 or 5R cells, approximately 2×10⁵ cellswere transfected with 0.25 μg of a reporter plasmid, 0.25 μg of EF1-lacZplasmid and 1 μg of either vector or effector plasmid. Cells wereharvested 40 to 45 hours after transfection. The amount of lysate usedfor luciferase assay was determined on the basis of β-galactosidaseactivity. The results shown are representative of one experiment carriedout in duplicate and averaged. Each experiment was repeated at leastthree times, with similar results.

Phoenix-Eco packaging cells were a kind gift of G. Nolan (StanfordUniversity).

Plasmids. A BLAST search of Genbank with the human IKK-1 cDNA sequencerevealed the presence of an EST clones encoding for a single,IKK-1-related cDNA. This clone was obtained from the UK HGMP and thecDNA insert was used to screen an adult human liver cDNA library.Positively hybridizing phage were isolated and both strands of thelargest insert obtained were sequenced by the dideoxy termination method(Sequenase, USB). IKK-2 coding sequences was amplified by PCR andinserted into vectors that allowed the in vitro and in vivo expressionof proteins fused to the VSV epitope. Rat IKK-1 was amplified by PCRfrom an EST clone and subcloned into the same vector. The plasmidsIgκ-luciferase and SRE-luciferase have been described previously(Courtois et al., 1997); HTLV-1 LTR-luciferase was a kind gift of P.Jalinot (Ecole Normale Suprieure de Lyon).

The plasmid Igκ2bsrH was constructed by ligating a 1.5 kb Hind III/BamHIfragment of the plasmid pSV2bsr (Izumi et al., 1991) with a 5.1 kb HindIII/BamHI fragment of the plasmid cx12lacZ-cB (Fiering et al., 1990)which contains 3 tandem copies of the NF-κB oligonucleotide derived fromthe Igκ sequence (TCAGAGGGGACTTTCCGAG) (SEQ ID NO:3) followed by aminimal IL-2 promoter.

A Tax expression vector, pCntax was constructed by inserting a BamHIfragment of the plasmid pUCwtax (Yamaoka et al., 1996) containing theentire coding sequence of Tax to the unique BamHI site of pCMV-Neo-Bamvector (Baker et al., 1990).

A 2.8 kb PCR product derived from genomic DNA of a blasticidinS-resistant 5R clone was obtained using primers located in theretroviral vector pCTV1 (Whitehead et al. 1995). This PCR product wasthen digested with SalI, subcloned into pBluescript for sequencing orinto the XhoI site of the CMV-hygro vector (a kind gift of F. Aurade,Institut Pasteur).

Reagents. LPS, PMA, poly (I-C), chloroquine and polybrene were fromSigma. Recombinant hIL-1β was from Biogen (Geneva, Switzerland).Recombinant TNF-α was from Genzyme. Blasticidin S was purchased fromICN. Absence of endotoxin contamination in all these reagents, exceptLPS, was checked with a polymyxin B assay (Shapiro and Dinarello, 1995).

Antisera. Rabbit antiserum against IκBα was a kind gift of J. DiDonatoand M. Karin (UCSD). Anti-VSV was mouse monoclonal P5D4. Anti-Tax wasmouse monoclonal MI73 (Mori et al., 1987). Anti-IKK-1 antibody was fromSanta Cruz. Anti NEMO rabbit polyclonal antiserum (serum 44106) wasraised against a TrpE fusion of a fragment encompassing amino acids30-329 of murine NEMO in the Path11 vector (Spindler et al., 1984).

Preparation of cell extracts. Cells were washed with PBS and resuspendedat 10⁶ cells/10 μl in hypotonic solution (10 mM Hepes, pH 7.8, 10 mMKCl, 2 mM MgCl₂, 1 mM DTT, 0.1 mM EDTA supplemented with a proteaseinhibitor cocktail (Boehringer)). After 10 minutes at 4_(i)C, NP40 wasadded to 1% and the cells centrifuged in a microfuge for 20 seconds. Thesupernatant, containing the cytoplasmic fraction, was recovered. Onevolume of 2× Laemmli buffer containing 20% β-Mercaptoethanol was addedand the sample was boiled for 5 minutes. The nuclear pellet was brieflywashed with hypotonic buffer and resuspended in 40 μl of extractionbuffer (50 mM Hepes, pH 7.8, 50 mM KCl, 350 mM NaCl, 0.1 mM EDTA, 1 mMDTT, 0.1 mM PMSF, 10% glycerol). After a 30 minutes incubation on ice,with occasional agitation, the DNA was pelleted by centrifuging at 14000rpm for 10 minutes. The supernatant, containing the nuclear fraction,was recovered and quickly frozen on dry ice. Samples were stored at −80°C.

Preparation of S100 extracts and gel filtration analysis. Fifty millionscells were washed in PBS and resuspended in 500 μl of 50 mM Tris pH 7.5,1 mM EGTA. Cells were lysed by thirty passages through a 26-gaugeneedle. After centrifugation for 10 minutes at 15000 rpm the supernatantwas recovered and complemented with 1 mM DTT, 0.025% Brij 35 and acocktail of proteases and phosphatases inhibitors. S100 were prepared bycentrifuging the cytoplasmic extracts for 30 minutes at 52000 rpm in aTLA 100.2 rotor (Beckman). After adding 10% glycerol, the S100 extractswere quickly frozen in dry ice and stored in liquid nitrogen. Gelfiltration chromatography was carried out on a Superose 6 column(Pharmacia) precalibrated with Aldolase (158 kD), Catalase (232 kD),Ferritin (440 kD) and Thyroglobulin (669 kD). Five hundred μl fractionswere recovered and directly analyzed by Western blotting orimmunoprecipitated with anti-NEMO. Silver staining of the fractions wasperformed with a Silver Stain Plus Kit (Biorad).

Western blot analysis. Proteins from cytoplasmic extracts werefractionated on 10% SDS-polyacrylamide gels, transferred onto Immobilonmembranes (Millipore), and blots were revealed with an enhancedchemiluminescence detection system (ECL, Amersham).

In vitro translation and crosslinking. Translations andco-immunoprecipitation experiments were performed as describedpreviously using TNT kits (Promega) (Kieran et al., 1990). Fordimerization experiments, translation reactions were diluted thirtytimes with phosphate buffered saline, treated with glutaraldehyde atroom temperature for 20 minutes, with 100 mM of Tris-HCl (pH 7.4) for 20minutes and subjected to immunoprecipitation after addition of an equalvolume of TNT buffer (NaCl 200 mM, Tris-HCl 20 mM pH 7.5, Triton X-1001% supplemented with protease and phosphatase inhibitors).Immunoprecipitations and kinase assays. Cytoplasmic extracts weresubjected to immunoprecipitation with anti-IKK-1 antibody, anti-NEMO orpre-immune serum in TNT buffer and collected on protein A-Sepharosebeads, which were then washed 3 times with TNT buffer and three timeswith kinase buffer (20 mM Hepes, 10 mM MgCl₂, 100 μM Na₃VO₄, 20 mMβ-glycerophosphate, 2 mM DTT, 50 mM NaCl, pH 7.5). Kinase reactions werefor 30 minutes at 30° C. using 5 μCi of [γ-³²P]-ATP and GST-IκBα (1-72)wild type or GST-IκBα (1-72) S32A/S36A mutant protein as substrates. Thereaction products were analyzed on 10% SDS-polyacrylamide gels andrevealed by autoradiography for three hours at room temperature.

Electrophoretic mobility shift assays. Five μg of nuclear extracts wereadded to 15 μl of binding buffer (10 mM Hepes pH 7.8, 100 mM NaCl, 1 mMEDTA, 10% glycerol final), 1 μg poly (dI-dC) and 0.5 ng ³²P-labeled κBprobe derived from the H-2 K^(b) promoter (Kieran et al., 1990), andincubated for 30 minutes at room temperature. Samples were run on a 5%polyacrylamide gel in 0.5×TBE.

Viral stocks and infection. T28 cells, a murine T cell hybridoma(Pyszniak et al., 1994) were cultured in Dulbecco's modified Eagle'smedium supplemented with 10% fetal bovine serum. Total mRNA fromexponentially growing T28 cells was used as template for cDNA synthesis,using random hexamer primers. Procedures for cDNA synthesis and cloningwere as described previously (Whitehead et al., 1995). The cDNA wasligated into pCTV1 (Whitehead et al., 1995), yielding 3.5×10⁶ cDNAclones. Complexities of the libraries were as follows: L35: 470,000clones (3.5 kb & up); L36: 600,000 clones (2.2-3.5 kb). The Phoenix-Ecopackaging cell line was used for transient transfection with DNA fromthe L35 or L36 libraries. To determine the virus titer on 5R cells, acDNA library (L20) cloned into the pCTV3 vector (Whitehead et al., 1995)which carries a hygromycin resistance gene was transfected by thecalcium phosphate method into Phoenix-Eco cells and the resultantsupernatants were titered by the appearance of hygromycin resistant 5Rcells. This library produced viral titers of 2-3×10⁵/ml. We producedviral supernatants for complementation experiments by transfectingapproximately 1.5×10⁷ Phoenix cells plated 24 hours before with 20 μg ofthe L35 or L36 library DNA in the presence of 25 μM chloroquine.Supernatants were recovered every 12 hours from 36 to 72 hours aftertransfection and either immediately used for infection of h12 cells orsnap frozen in dry ice and stored at −80° C. Approximately 10⁶ h12 cellswere plated 12 to 15 hours before infection on a 100 mm petri dish andexposed to 3 ml of viral supernatant in the presence of 3 ml ofconditioned medium and 10 μg/ml of polybrene. Twelve hours afterstarting the infection, the viral supernatant was removed and cells werecultured for an additional 24 hours in normal growth medium. BlasticidinS was added to a final concentration of 10 μg/ml 36 hours afterinfection. The selection medium was replaced at least every 5 days andthe resultant cell clones were isolated with cloning cylinders. We useda total of 30×10⁶ h12 cells for infection with virus stock obtainedusing 20 μg of L35 or L36 library DNA and finally isolated similarnumber of independent cell clones for the two cDNA libraries.

Results Characterization of the Mutant Cell Line 5R

Spontaneous flat revertant cells were isolated from M319-5 cells, aclone of Rat-1 fibroblasts transformed by a mutant Tax protein competentto activate NF-κB, but unable to stimulate HTLV-1 long terminal repeat(LTR)-directed transcription (Yamaoka et al., 1996). All of them exceptone (clone 5R) had lost Tax expression. 5R cells express Tax at a levelcomparable with the parental cells (FIG. 1A, lane 3), but are defectivein Tax-induced NF-κB DNA-binding activity (FIG. 1B, lane 3). Stableexpression of wild-type Tax failed to re-transform 5R cells, whileforced expression of constitutively active c-Ha-Ras or v-Src proteintransformed 5R cells as efficiently as the parental Rat-1 cells.Transient expression of wild-type Tax fully activated HTLV-1LTR-directed, but not NF-κB dependent transcription in 5R cells (FIGS.1C, D). On the other hand, transient expression of RelA or activatedc-Ha-Ras strongly stimulated NF-κB- or Serum ResponsiveElement-dependent transcription respectively, in 5R as well as in Rat-1cells (FIG. 1D and data not shown). These results suggest that 5R cellscarry a mutation(s) which abrogates Tax-mediated NF-κB activation.

The phenotype of the mutation was next analyzed by somatic cellhybridization. Since 5R cells express Tax, they are expected to restoreTax-induced NF-κB activity after hybridization with parental cells ifthe mutation is recessive. Hybridization of 5R cells with Rat-1 cellscarrying an integrated NF-κB-dependent reporter gene induced a strongtranscriptional activity when compared with the control hybridization(FIG. 1E). We also established a pooled population of stable hybridsbetween 5R and Rat-1 cells and found that they exhibited a transformedphenotype (data not shown) and contained high NF-κB DNA-binding activity(FIG. 1B, lane 4). These results indicate that the phenotype of themutation in 5R cells is recessive and therefore should be amenable togenetic complementation.

Rat-1 cells normally activate NF-κB in response to diverse externalstimuli, including tumor necrosis factor-α (TNF-α), interleukin-1(IL-1), lipopolysaccharide (LPS) or double stranded RNA (dsRNA).Interestingly, none of these stimuli was able to induce NF-κBDNA-binding activity in 5R cells (FIG. 2A). This result was furtherconfirmed by transient transfection with an NF-κB-dependent reportergene (FIG. 2B). In order to identify the step at which NF-κB signalingis affected, the levels of IκB proteins were examined in cellsstimulated with LPS. As shown in FIG. 2C, LPS stimulation led to acomplete loss of IκBα and of IκBβ in Rat-1 cells followed byre-appearance of IκBα 60 minutes after stimulation. In contrast, thelevels of the two IκB proteins in 5R cells were virtually unaffected byLPS treatment. Taken together, one can conclude that 5R cells carry arecessive mutation(s) at a converging regulatory step leading toinducible degradation of IκB proteins. Finally, the possibility that 5Rcells might be defective in one of the functional IκB kinases wasaddressed. Stable transfection of 5R cells with plasmids encoding eitherIKK-1 or IKK-2 did not restore NF-κB activity.

Molecular Cloning of NEMO

For complementation experiments, a selection system was established bypreparing sublines of 5R cells capable of expressing an NF-κB-dependentinducible drug resistance gene. A conditional drug resistance gene,pIgκlbsμH contains both a hygromycin resistance gene under the controlof the HSV1 thymidine kinase gene promoter, and the blasticidindeaminase gene (Izumi et al., 1991) linked to a minimal IL-2 promoterfollowing three repeats of the immunoglobulin κ light chain NF-cBbinding site. Stable transfection of the parental Tax transformed cellswith this construct using hygromycin selection followed by selectionwith blasticidin S resulted in numerous surviving colonies, whereas nonecould be observed for 5R cells. Hygromycin-resistant 5R clones weretested for survival in the presence of blasticidin S following simpleco-culture or hybridization with normal Rat-1 cells. One of the 5Rclones, h12, was chosen at random for further experiments as being ableto survive a high dose of blasticidin S selection after thehybridization, but showing absolutely no survival at a low concentrationof the drug without the hybridization step. A high NF-κB DNA-bindingactivity was detected in stable h12/Rat-1 hybrids, a result ofactivation by Tax following complementation of the defect of h12 cells.

Approximately 30×10⁶ h12 cells were infected with retroviruses carryinga cDNA expression library derived from the T28 murine T cell hybridomacell line (Whitehead et al., 1995). Viral supernatants were produced bytransient transfection of Phoenix cells with the retroviral constructsgiving titers in the range of 2×10⁵-3×10⁵/ml. Selection with blasticidinS was started 36 hours after viral infection. In 20 to 30 days, a totalof more than 40 independent clones were obtained and 20 were tested fortheir NF-κB DNA-binding activity. All clones except one contained highlevels of DNA-binding activity and clearly showed a transformedphenotype (FIG. 4B, lanes 5-6). Polymerase chain reaction-mediatedamplification of genomic DNAs from seven clones resulted in aprovirus-derived specific band with a size of 3.2 kb, while 33 otherclones carried a 2.8 kb insert. Southern blot analysis of the 3.2 kbinsert showed cross hybridization with the 2.8 kb fragment. Sequencinganalysis of the amplified 2.8 kb cDNA showed that it contained an openreading frame predicted to encode a previously unknown 48 kDapolypeptide, which we have named NEMO (NF-κB Essential MOdulator) (FIG.3). This molecule is acidic (pI 5.66) and unusually rich in glutamicacid and glutamine (13% each). In addition, it contains a putativeleucine zipper motif (amino acids 315 to 342). To characterize itsfunction, Rat-1 or 5R cells were transfected with a mammalian expressionvector capable of expressing NEMO. Cotransfection of 5R cells with avery small amount of NEMO and an NF-κB-dependent reporter gene resultedin a strong reporter gene activation by endogenous Tax, whereas itsoverexpression in Rat-1 cells barely activated the reporter construct(FIG. 4A). Rat-1 or 5R cells stably expressing NEMO were thenestablished. As expected, stable expression of NEMO (under the controlof the strong CMV promoter) in wild-type Rat-1 cells did not give riseto detectable NF-κB activity (FIG. 4B, lane 2). On the other hand, twopooled populations derived from NEMO-transformed 5R cells and twoisolated clones showed high levels of NF-κB DNA-binding activity (lanes7-10), indicating that stable NEMO expression can complement the defectin 5R cells.

A polyclonal antibody was raised against the region encompassing aminoacids 60-329 of NEMO and used to analyze its expression in 5R cells.Whereas the protein could be readily detected as a single 48% band inRat1 cytoplasmic extracts (FIG. 4C), no NEMO band could be observed in5R cells. In addition, no truncated form of the protein could bedetected. Thus, the defective phenotype of 5R cells results from theabsence of the NEMO protein.

Complementation of the 1.3E2 Mutant Cell Line by NEMO

The characterization of another mutant cell line, the 70Z/3-derivedmutant 1.3E2, was recently reported that exhibits a defect in NF-κBactivation (Courtois et al., 1997). In this cell line NF-κB is notactivated in response to a large set of stimuli, among them LPS, IL-1,PMA, dsRNA or TNF. This is due to a lack of IκBα, IκB and IκBεdegradation. Since phosphorylation of IκBα on Ser 32 and 36 is notobserved after stimulation, a converging step preceding the IκBphosphorylation step or the phosphorylation step itself was proposed tobe deficient in 1.3E2.

Since the 1.3E2 phenotype shares many similarities with the 5Rphenotype, it was tested whether NEMO could complement 1.3E2.Strikingly, as shown in FIG. 5A, transient transfection of 1.3E2 with avector expressing NEMO allowed the recovery of a wild-type NF-κBactivation level after LPS stimulation. Such an effect was clearlystimulus-specific, indicating that NEMO overexpression by itself wasunable to activate NF-κB. Complemention was also observed in the case oftwo other stimuli, IL-1 and PMA, although with less efficiency in thelatter case.

1.3E2 cells stably expressing NEMO (1.3E2N) were also prepared andtested for complementation. A mobility shift experiment presented inFIG. 5B confirmed the results of the transient transfection experimentsdescribed above. NF-κB activation in response to LPS, IL-1 or PMA wasfound to be similar in wild-type 70Z/3 and 1.3E2N. Moreover, animmunoblot analysis revealed that NEMO is undetectable in 1.3E2 cells(FIG. 5C). These results demonstrate that, as for 5R cells, thephenotype of the 1.3E2 mutant cell line is due to the absence of NEMO.

NEMO is Part of the IκB Kinase Complex

Since NEMO appears to be critically involved in NF-κB activation by alarge set of stimuli and complements cells defective in IκBphosphorylation, an attractive possibility would be that it constitutesa subunit of the 600-800 kD kinase complex that phosphorylates IκB.Therefore, it was investigated whether NEMO is associated with theinducible IκB kinase activity (FIG. 6). To demonstrate this point immunecomplex kinase assays were conducted on Rat-1 or 5R cells. The antiserumagainst NEMO immunoprecipitated a specific endogenous IκBα kinaseactivity from wild type cells stimulated with TNF-α. Absense of kinaseactivity in NEMO-immunoprecipitates from 5R cells and lack ofphosphorylation of a mutant IκBα polypeptide (S32A, S36A) establishedthe specificity of the antiserum and kinase activity, respectively.Thus, NEMO is associated with an inducible endogenous IκBα kinaseactivity. As reported previously, an anti-IKK-1 antibody brought down aspecific IκBα kinase activity from wild type cells stimulated with TNF-αfor 5 minutes. Interestingly, no inducible IκBα kinase activity wasobserved in IKK-1 precipitates from 5R cell extracts.

In order to confirm that NEMO is an integral part of the IκB kinasecomplex, and to determine whether it is stably associated with it beforestimulation, S100 extracts were prepared from Rat-1 cells andfractionated on a Superose 6 gel filtration column. Elution of the IκBkinase, monitored with an anti-IKK-1 antibody, was mostly observed infractions containing proteins of 600 to 800 kD, as previously reported(FIG. 7A). When NEMO elution was examined, an identical profile wasobtained. Immunoprecitation of the NEMO-containing fractions with ananti-NEMO antibody allowed us to co-immunoprecipitate IKK-1 (FIG. 7B).NEMO is therefore a stable component of the 600-800 kDa IκB kinasecomplex.

Quite remarkably, when 5R extracts were analyzed with the IKK-1antibody, the elution peak appeared shifted toward fractions containingproteins of 300-450 kD instead of 600-800 kD (FIG. 7A). Since theoverall elution profile, as checked either by silver staining (FIG. 7A,top panel) or by Western blotting against RelA (FIG. 7A, bottom panel)or p105 (data not shown), was identical between Rat-1 and 5R, thisobservation demonstrated the requirement of NEMO for building a highmolecular weight IκB kinase complex. Moreover, the absence of IκB kinaseactivity in 5R cells after stimulation (see above) indicates that thelower molecular weight kinase complex is refractory to activation.

NEMO can Form Homodimers and Interacts Directly with IKK-2

The presence of a leucine zipper-like motif in NEMO led us to askwhether this molecule could dimerize. Glutaraldehyde crosslinkingexperiments (FIG. 7C) demonstrated that NEMO was indeed able to formhomodimers.

Since NEMO is part of the IκB kinase complex, direct interactions withknown components of the complex were examined, namely the two catalyticsubunits IKK-1 and IKK-2. An in vitro analysis was conducted using³⁵S-labeled proteins translated in wheat germ extracts (WGE). Afterco-translation of VSV-IKK-2 and NEMO, followed by anti-VSVimmunoprecipitation, NEMO was readily detected in the immunoprecipitate(FIG. 7D). The converse experiment, using NEMO plus VSV-IKK-2 andimmunoprecipitating with anti-NEMO allowed the detection of VSV-IKK-2 inthe immunoprecipitate. Interestingly, such an interaction could barelybe observed with IKK-1, suggesting a potential functional divergencebetween the two IKKs.

One approach aimed at identifying components of the NF-κB signalingpathway which has not been widely used so far is to generate mutant celllines which are unresponsive to one or several NF-κB activating signals,and to try and complement these cell lines with genomic or cDNAlibraries (Ting et al., 1996). Here is used a spontaneous mutant (called5R) of a HTLV-1 transformed Rat-1 fibroblastic cell line, which had lostits transformed morphology. This mutation was accompanied bydisappearance of Tax-induced NF-κB activity, as measured by bandshiftand transactivation assays. In addition, LPS-, IL-1-dsRNA- orTNF-induced NF-κB DNA-binding activity could not be observed in the 5Rcell line. However other signaling pathways seemed to be stillfunctional. Importantly, cell fusion experiments demonstrated that themutation was recessive. All these observations prompted an attempt tocomplement this cell line. The selection was based on introduction intothese cells, prior to complementation, of a gene encoding resistance tothe antibiotic blasticidin S driven by multimerized NF-κB binding sites.Only the complemented cells would be expected to become resistant toblasticidin S treatment, a result of transactivation of the blasticidinS resistance gene by endogenous Tax. More than forty independentblasticidin S-resistant clones were isolated, and bandshift analysisdemonstrated the presence of a p50/relA complex in 19 analyzed clonesout of 20, with an intensity similar to that observed followingstimulation of wild-type Rat-1 cells with LPS or TNF. PCR amplificationof DNA from 40 independent clones using primers localized in theflanking regions of the retroviral vector yielded two cross-hybridizingfragments, of 2.8 and 3.2 kb. Sequencing of the amplified cDNA revealedthat the 2.8 kb insert contains an open reading frame encoding apreviously undescribed 412 amino acid protein, that we called NEMO. Thisprotein is acidic (pI 5.66), unusually rich in glutamic acid andglutamine (13% each) and also contains a putative leucine zipper motif(amino acids 315 to 342).

Transfection of NEMO complemented the mutation in 5R cells. This led tothe conclusion that NEMO is necessary for activation of NF-κB by Tax.However the presence of endogenous Tax in the 5R cell line precluded theanalysis of NEMO involvement in other NF-κB activation pathways. Thisproblem was circumvented by the use of 1.3E2, another mutant cell linethat we previously characterized (Courtois et al., 1997). NF-κBactivation, degradation of the three known IκB inhibitors, as well asinduced phosphorylation of IκBα could not be observed following PMA,LPS, IL-1 or dsRNA treatment of this cell line. NEMO cDNA was stablyintroduced into 1.3E2 and it was observed that NF-κB activation by atleast three of these stimuli (LPS, PMA and IL-1) was restored. Thereforethe NEMO protein is involved in the response to at least four NF-κBactivating stimuli.

An interesting conclusion one can draw from complementation of the 5Rcells, which regain a transformed phenotype when stably transfected withNEMO, is that NF-κB activity seems to be required for celltransformation by Tax (at least in this cell system). There have beenconflicting data in the literature concerning the actual involvement ofNF-κB in Tax-induced transformation (Kitajima et al., 1992; Smith andGreene, 1991; Yamaoka et al., 1996).

The next question concerned the actual function of NEMO. Since thismolecule appears to be involved in all tested NF-κB activating pathways,an obvious possibility was that it constituted one subunit of the highmolecular weight IκB kinase complex. Three arguments are in favor ofthis hypothesis: First, immunoprecipitation of NEMO from Rat-1 cellspulled down a bona fide IκBα kinase activity, specific for the 2N-terminal serines. Second, NEMO elutes as a 600-800 kDa peak from a gelfiltration column performed on extracts from unstimulated Rat-1 cells,as does IKK-1. Third, immunoprecipitation of NEMO from Rat-1 fractionsranging from 600 to 800 KDa brings down IKK-1.

The possible interaction of NEMO with the 2 catalytic subunits of thecomplex was tested, IKK-1 and IKK-2. In vitro cotranslation of IKK-2 andNEMO in wheat germ extract followed by immunoprecipitation demonstratedthat the two proteins could interact with each other. In contrast aninteraction between NEMO and IKK-1 could barely be detected under theseconditions. NEMO can also form homodimers.

IKK-1 can be detected in a 300-450 kD complex in 5R cells, thereforeindicating that NEMO is required for the formation of a 600-800 kDafunctional IKK complex, and probably plays a role as a structuralcomponent of this complex.

It was unexpected that two independently isolated mutant cell linescould be complemented by the same cDNA. The selection forLPS-unresponsive derivatives of 70Z/3 yielded several types of mutantcell lines, but only 1.3E2 was also unresponsive to other NF-κBactivating stimuli, and the fact that it grows faster than the wild-type70Z/3 probably facilitated its isolation. In Tax transformed Rat-1cells, 5R was the only NF-κB defective cellular revertant which could beisolated. Mutating the nemo gene might be the only means of knocking outNF-κB activation by a single gene mutation.

The following references are cited herein, and should be considered tobe incorporated herein by reference in their entireties:

REFERENCES

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Having now fully described the invention, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the inventionas set forth herein.

1-11. (canceled)
 12. A purified DNA sequence which encodes a purifiedmodulator of NF-κB, or a fragment thereof which binds to a component ofan IκB kinase, selected from the group consisting of: (A) the DNAsequence of SEQ ID NO: 2; (B) DNA sequences that hybridize to the DNAsequence of (A) under standard hybridization conditions; and (C) DNAsequences that encode a purified modulator of NF-κB, comprising amaterial selected from the group consisting of a protein, activefragments thereof, agonists thereof, mimics thereof and combinationsthereof, said modulator having the following characteristics: a) anapparent molecular weight of approximately 48 kD; b) a pI ofapproximately 5.66: c) containing a leucine zipper motif; and d) bindingto a kinase involved in the activation of NF-κB.
 13. A recombinant DNAmolecule comprising the purified DNA sequence of claim
 12. 14. Therecombinant DNA molecule of claim 13, wherein said DNA sequence isoperatively linked to an expression control sequence.
 15. Therecombinant DNA molecule of claim 14, wherein said expression controlsequence is selected from the group consisting of the early or latepromoters of CMV, the lac system, the trp system, the TAC system, theTRC system, the major operator and promoter regions of phage λ, thecontrol regions of fd coat protein, the promoter for 3-phosphoglyceratekinase, the promoters of acid phosphatase and the promoters of the yeastα-mating factors.
 16. A probe capable of screening for a DNA sequenceencoding a modulator of NF-κB in alternate species, wherein said probeis a fragment of the purified DNA sequence of claim 12 which binds tothe DNA sequence encoding a modulator of NF-κB in alternate speciesunder standard hybridization conditions.
 17. A unicellular hosttransformed with the recombinant DNA molecule of claim
 14. 18. Theunicellular host of claim 17 wherein the unicellular host is selectedfrom the group consisting of E. coli, Pseudomonas, Bacillus,Streptomyces, yeasts, CHO, R1.1, B-W, L-M, COS 1, COS 7, BSC1, BSC40,and BMT10 cells, plant cells, insect cells, and human cells in tissueculture.
 19. A method for producing a purified modulator of NF-κB,comprising a material selected from the group consisting of a protein,active fragments thereof, agonists thereof, mimics thereof, andcombinations thereof, said modulator having the followingcharacteristics: a) an apparent molecular weight of approximately 48 kD;b) a pI of approximately 5.66; c) containing a leucine zipper motif; andd) binding to a kinase involved in the activation of NF-κB, wherein saidmethod comprises: (a) culturing the unicellular host of claim 17; and(b) isolating the modulator of NF-κB from said cultured unicellularhost. 20-30. (canceled)
 31. A recombinant virus transformed with the DNAmolecule of claim
 12. 32-33. (canceled)
 34. An antisense nucleic acidagainst an mRNA encoding a purified modulator of NF-κB, comprising amaterial selected from the group consisting of a protein, activefragments thereof, agonists thereof, mimics thereof, and combinationsthereof, said modulator having the following characteristics: a) anapparent molecular weight of approximately 48 kD; b) a pI ofapproximately 5.66; c) containing a leucine zipper motif; and d) bindingto a kinase involved in the activation of NF-κB, wherein said antisensenucleic acid comprises a nucleic acid sequence hybridizing to said mRNA.35. The antisense nucleic acid of claim 34 which is RNA.
 36. Theantisense nucleic acid of claim 35 which is DNA.
 37. The antisensenucleic acid of claim 35 which binds to the initiation codon of saidmRNA.
 38. A recombinant DNA molecule having a DNA sequence which, ontranscription, produces an antisense ribonucleic acid against an mRNAencoding a purified modulator of NF-κB, comprising a material selectedfrom the group consisting of a protein, active fragments thereof,agonists thereof, mimics thereof, and combinations thereof, saidmodulator having the following characteristics: a) an apparent molecularweight of approximately 48 kD; b) a pI of approximately 5.66; c)containing a leucine zipper motif; and d) binding to a kinase involvedin the activation of NF-κB, wherein said antisense ribonucleic acidcomprising an nucleic acid sequence capable of hybridizing to said mRNA.39. A cell line transfected with the recombinant DNA molecule of claim38.
 40. A method for creating a cell line which exhibits reducedtranscriptional activity, comprising transfecting an NF-κB producingcell line with the recombinant DNA molecule of claim
 38. 41-50.(canceled)